Herpes simplex virus type 1 (HSV-1) causes clinically significant acute, and life-long latent infections which reactivate periodically causing virus shedding and recurrent disease in a large percentage of the population worldwide. Although reactivated virus is recognized as the primary source of infection in spread to uninfected individuals, little is known about the molecular events that initiate reactivation. Because reactivation is an experimentally approachable process at the molecular level, Project 3 will address three aspects central to the initiation of reactivation. Both stress and expression of the HSV-1 regulatory protein, ICPO, have been shown to induce reactivation from latency. In Specific Aim 1, the cellular stress-induced and cell cycle-associated proteins whose expression is induced or repressed by both stress and ICPO will be identified during productive infection of Vero cells and reactivation from neuronal latency using focused microarray RT-PCR and Western blot analysis. The roles of these proteins in reactivation will be ascertained by RNA silencing of proteins induced by stress and ICPO, and over-expressing proteins repressed by stress and ICPO. The viral proteins expressed first in latently infected neurons in response to stress likely play central roles in initiating the viral replication process during the early stages of reactivation. In Specific Aim 2, these viral proteins will be identified by a combination of microarray, RT-PCR, and Western blot analysis. The elements in the promoters of viral genes that respond to stress will be identified and mutated in the viral genome. The resulting mutant viruses will be tested for their ability to reactivate from latency following stress. Among the cellular proteins whose expression is altered in response to stress and the expression of ICPO are cdks and their cyclin partners. In Specific Aim 3, the cdks whose localization, expression levels, or activities are altered in response to stress will be identified. Ad vectors expressing siRNAs that silence cdks that are overexpressed, and Ad vectors that overexpress cdks that are down-regulated in response to stress will be tested for their ability to alter the reactivation efficacy of wt virus in latently-infected TG cell cultures in response to stress. Collectively, these studies will provide new targets for antiviral drug development and suggest novel strategies for inhibiting HSV-1 reactivation.